The present invention is concerned with a novel vascular endothelial growth factor (VEGF) herein designated xe2x80x9cVEGF-Xxe2x80x9d, and characterisation of the nucleic acid and amino acid sequences of VEGF-X.
Angiogenesis involves formation and proliferation of new blood vessels, and is an essential physiological process for normal growth and development of tissues in, for example, embryonic development, tissue regeneration and organ and tissue repair. Angiogenesis also features in the growth of human cancers which require continuous stimulation of blood vessel growth. Abnormal angiogenesis is associated with other diseases such as rheumatoid arthritis psoriasis and diabetic retinopathy.
Capillary vessels consist of endothelial cells which carry the genetic information necessary to proliferate to for capillary networks. Angiogenic molecules which can initiate this process have previously been characterised. A highly selective mitogen for vascular enothelial cells is vascular endothelial growth factor (VEGF) (Ferrara et al., xe2x80x9cVascular Endothelial Growth Factor: Basic Biology and Clinical Implicationsxe2x80x9d. Regulation of angiogenesis, by I. D. Goldberg and E. M. Rosen 1997 Birkhauser Verlag Basle/Switzerland). VEGF is a potent vasoactive protein which is comprised of a glycosylated cationic 46-49 kd dimer having two 24 kd subunits. It is inactivated by sulfhydryl reducing agents and is resistant to acidic pH and to heating and binds to immobilised heparin. VEGF-A has four different forms of 121, 165, 189 and 206 amino acids respectively due to alternative splicing. VEGF121 and VEGF165 are soluble and are capable of promoting angiogenesis, whereas VEGF189 and VEGF206 are bound to heparin containing proteoglycans in the cell surface. The temporal and spatial expression of VEGF has been correlated with physiological proliferation of the blood vessels (Gajdusek, C. M., and Carbon, S. J., Cell Physiol., 139:570-579, (1989)); McNeil, P. L., Muthukrishnan, L., Warder, E., D""Amore, P. A., J. Cell. Biol., 109:811-822, (1989)). Its high affinity binding sites are localized only on endothelial cells in tissue sections (Jakeman, L. B., et al., Clin. Invest. 89:244-253 (1989)). The growth factor can be isolated from pituitary cells and several tumor cell lines, and has been implicated in some human gliomas (Plate, K. H. Nature 359:845-848, (1992)). The inhibition of VEGF function by anti-VEGF monoclonal antibodies was shown to inhibit tumor growth in immune-deficient mice (Kim, K. J., Nature 362:841-844, (1993)).
VEGF proteins have been described in the following patents and applications all of which are hereby incorporated by reference EP-0,506,477, WO-95/24473, WO-98/28621, WO-90/13649, EP-0,476,983, EP-0,550,296, WO-90/13649, WO-96/26736, WO-96/27007, WO-98/49300, WO-98/36075, WO-90/11084, WO-98/24811, WO-98/10071, WO-98/07832, WO-98/02543, WO-97/05250, WO-91/02058, WO-96/39421, WO-96/39515, WO-98/16551.
The present inventors have now identified a further vascular endothelial growth factor, designated herein as xe2x80x9cVEGF-Xxe2x80x9d, and the nucleic acid sequence encoding it, which has potentially significant benefits for the treatment of tumours and other conditions mediated by inappropriate angiogenic activity.
In the present application, there is provided a novel vascular endothelial growth factor, herein designated xe2x80x9cVEGF-Xxe2x80x9d, nucleic acid molecules encoding said growth factor, an expression vector comprising said nucleic acid molecule, a host cell transformed with said vector and compounds which inhibit or enhance angiogenesis. Also provided is the sequence of a CUB domain present in the sequence of VEGF-X which domain itself prevents angiogenesis and which is used to treat diseases associated with inappropriate vascularisation or angiogenesis.
Therefore, according to a first aspect of the present invention there is provided a nucleic acid molecule encoding a VEGF-X protein or a functional equivalent, fragment, derivative or bioprecursor thereof, said protein comprising the amino acid sequence from position 23 to 345 of the amino acid sequence illustrated in FIG. 10. Alternatively, the nucleic acid molecule of the invention encodes the complete sequence identified in FIG. 10 and which advantageously includes a signal peptide to express said protein extracellularly. Preferably, the nucleic acid molecule is a DNA and even more preferably a cDNA molecule. Preferably, the nucleic acid molecule comprises the nucleotide sequence from position 257 to 1291 of the nucleotide sequence illustrated in FIG. 9. In a preferred embodiment the nucleic acid is of mammalian origin and even more preferably of human origin.
In accordance with the present invention a functional equivalent should be taken to mean a protein, or a sequence of amino acids that have similar function to the VEGF-X protein of the invention.
Also provided by this aspect of the present invention is a nucleic acid molecule such as an antisense molecule capable of hybridising to the nucleic acid molecules according to the invention under high stringency conditions, which conditions would be well known to those skilled in the art.
Stringency of hybridisation as used herein refers to conditions under which polynucleic acids are stable. The stability of hybrids is reflected in the melting temperature (Tm) of the hybrids. Tm can be approximated by the formula:
81.5xc2x0 C.+16.6(log10[Na+]+0.41 (%GandC)xe2x88x92600/1
wherein 1 is the length of the hybrids in nucleotides. Tm decreases approximately by 1-1.5xc2x0 C. with every 1% decrease in sequence homology.
The term xe2x80x9cstringencyxe2x80x9d refers to the hybridisation conditions wherein a single-stranded nucleic acid joins with a complementary strand when the purine or pyrimidine bases therein pair with their corresponding base by hydrogen bonding. High stringency conditions favour homologous base pairing whereas low stringency conditions favour non-homologous base pairing.
xe2x80x9cLow stringencyxe2x80x9d conditions comprise, for example, a temperature of about 37xc2x0 C. or less, a formamide concentration of less than about 50%, and a moderate to low salt (SSC) concentration; or, alternatively, a temperature of about 50xc2x0 C. or less, and a moderate to high salt (SSPE) concentration, for example 1M NaCl.
xe2x80x9cHigh stringencyxe2x80x9d conditions comprise, for example, a temperature of about 42xc2x0 C. or less, a formamide concentration of less than about 20%, and a low salt (SSC) concentration; or, alternatively, a temperature of about 65xc2x0 C., or less, and a low salt (SSPE) concentration. For example, high stringency conditions comprise hybridization in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65xc2x0 C. (Ausubel, F. M. et al. Current Protocols in Molecular Biology, Vol. I, 1989; Green Inc. New York, at 2.10.3).
xe2x80x9cSSCxe2x80x9d comprises a hybridization and wash solution. A stock 20xc3x97SSC solution contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0.
xe2x80x9cSSPExe2x80x9d comprises a hybridization and wash solution. A 1xc3x97SSPE solution contains 180 mM NaCl, 9 mM Na2HPO4 and 1 mM EDTA, pH 7.4.
The nucleic acid capable of hybridising to nucleic acid molecules according to the invention will generally be at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the nucleotide sequences according to the invention.
The antisense molecule capable of hybridising to the nucleic acid according to the invention may be used as a probe or as a medicament or may be included in a pharmaceutical composition with a pharmaceutically acceptable carrier, diluent or excipient therefor.
The term xe2x80x9chomologousxe2x80x9d describes the relationship between different nucleic acid molecules or amino acid sequences wherein said sequences or molecules are related by partial identity or similarity at one or more blocks or regions within said molecules or sequences.
The present invention also comprises within its scope proteins or polypeptides encoded by the nucleic acid molecules according to the invention or a functional equivalent, derivative or bioprecursor thereof.
Therefore, according to a further aspect of the present invention, there is provided a VEGF-X protein, or a functional equivalent, derivative or bioprecursor thereof, comprising an amino acid sequence from position 23 to 345 of the sequence as illustrated in FIG. 10, or alternatively which amino acid sequence comprises the complete sequence of FIG. 10. A further aspect of the invention comprises a VEGF-X protein, or a functional equivalent, derivative or bioprecusor thereof, encoded by a nucleic acid molecule according to the invention. Preferably, the VEGF-X protein encoded by said nucleic acid molecule. comprises the sequence from position 23 to 345 of the amino acid sequence as illustrated in FIG. 10, or which sequence alternatively comprises the sequence of amino acids of FIG. 10.
The DNA molecules according to the invention may, advantageously, be included in a suitable expression vector to express VEGF-X encoded therefrom in a suitable host. Incorporation of cloned DNA into a suitable expression vector for subsequent transformation of said cell and subsequent selection of the transformed cells is well known to those skilled in the art as provided in Sambrook et al. (1989), molecular cloning, a laboratory manual, Cold Spring Harbour Laboratory Press.
An expression vector according to the invention includes a vector having a nucleic acid according to the invention operably linked to regulatory sequences, such as promoter regions, that are capable of effecting expression of said DNA fragments. The term xe2x80x9coperably linkedxe2x80x9d refers to a juxta position wherein the components described are in a relationship permitting them to function in their intended manner. Such vectors may be transformed into a suitable host cell to provide for expression of a polypeptide according to the invention. Thus, in a further aspect, the invention provides a process for preparing polypeptides according to the invention which comprises cultivating a host cell, transformed or transfected with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptides, and recovering the expressed polypeptides.
The vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, and optionally a promoter for the expression of said nucleotide and optionally a regulator of the promoter.
The vectors may contain one or more selectable markers, such as, for example, ampicillin resistance.
Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. For example, a bacterial expression vector may include a promoter such as the lac promoter and for translation initiation the Shine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryotic expression vector may include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors may be obtained commercially or assembled from the sequences described by methods well known in the art.
Nucleic acid molecules according to the invention may be inserted into the vectors described in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense nucleic acids may be produced by synthetic means.
In accordance with the present invention, a defined nucleic acid includes not only the identical nucleic acid but also any minor base variations including in particular, substitutions in cases which result in a synonymous codon (a different codon specifying the same amino acid residue) due to the degenerate code in conservative amino acid substitutions. The term xe2x80x9cnucleic acid sequencexe2x80x9d also includes the complementary sequence to any single stranded sequence given regarding base variations.
The present invention also advantageously provides nucleic acid sequences of at least approximately 10 contiguous nucleotides of a nucleic acid according to the invention and preferably from 10 to 50 nucleotides even more preferably, the nucleic acid sequence comprise the sequences illustrated in FIG. 3. These sequences may, advantageously be used as probes or primers to initiate replication, or the like. Such nucleic acid sequences may be produced according to techniques well known in the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or the like for detecting the presence of a nucleic acid according to the invention. These tests generally comprise contacting the probe with the sample under hybridising conditions and detecting for the presence of any duplex or triplex formation between the probe and any nucleic acid in the sample.
The nucleic acid sequences according to this aspect of the present invention comprise the sequences of nucleotides illustrated in FIGS. 3 and 5.
According to the present invention these probes may be anchored to a solid support. Preferably, they are present on an array so that multiple probes can simultaneously hybridize to a single biological sample. The probes can be spotted onto the array or synthesised In situ on the array. (See Lockhart et al., Nature Biotechnology, vol. 14, December 1996 xe2x80x9cExpression monitoring by hybridisation to high density oligonucleotide arraysxe2x80x9d. A single array can contain more than 100, 500 or even 1,000 different probes in discrete locations.
The nucleic acid sequences, according to the invention may be produced using such recombinant or synthetic means, such as for example using PCR cloning mechanisms which generally involve making a pair of primers, which may be from approximately 10 to 50 nucleotides to a region of the gene which is desired to be cloned, bringing the primers into contact with mRNA, cDNA, or genomic DNA from a human cell, performing a polymerase chain reaction under conditions which brings about amplification of the desired region, isolating the amplified region or fragment and recovering the amplified DNA. Generally, such techniques are well known in the art, such as described in Sambrook et al. (Molecular Cloning: a Laboratory Manual, 1989).
The nucleic acids or oligonucleotides according to the invention may carry a revealing label. Suitable labels include radioisotopes such as 32P or 35S, enzyme labels or other protein labels such as biotin or fluorescent markers. Such labels may be added to the nucleic acids or oligonucleotides of the invention and may be detected using known techniques per se.
Advantageously, human allelic variants or polymorphisms of the DNA molecule according to the invention may be identified by, for example, probing cDNA or genomic libraries from a range of individuals, for example, from different populations. Furthermore, nucleic acids and probes according to the invention may be used to sequence genomic DNA from patients using techniques well known in the art, such as the Sanger Dideoxy chain termination method, which may, advantageously, ascertain any predisposition of a patient to certain disorders associated with a growth factor according to the invention.
The protein according to the invention includes all possible amino acid variants encoded by the nucleic acid molecule according to the invention including a polypeptide encoded by said molecule and having conservative amino acid changes. Conservative amino acid substitution refers to a replacement of one or more amino acids in a protein as identified in Table 1. Proteins or polypeptides according to the invention further include variants of such sequences, including naturally occurring allelic variants which are substantially homologous to said proteins or polypeptides. In this context, substantial homology is regarded as a sequence which has at least 70%, preferably 80 or 90% and preferably 95% amino acid homology with the proteins or polypeptides encoded by the nucleic acid molecules according to the invention. The protein according to the invention may be recombinant, synthetic or naturally occurring, but is preferably recombinant.
The nucleic acid or protein according to the invention may be used as a medicament or in the preparation of a medicament for treating cancer or other diseases or conditions associated with expression of VEGF-X protein.
Advantageously, the nucleic acid molecule or the protein according to the invention may be provided in a pharmaceutical composition together with a pharmacologically acceptable carrier, diluent or excipient therefor.
The present invention is further directed to inhibiting VEGF-X in vivo by the use of antisense technology. Antisense technology can be used to control gene expression through triple-helix formation of antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5xe2x80x2 coding portion or the mature DNA sequence, which encodes for the protein of the present invention, is used to design an antisense RNA oligonucleotide of from 10 to 50 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple-helixxe2x80x94see Lee et al. Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991), thereby preventing transcription and the production of VEGF-X. The antisense RNA oligonucleotide hybridises to the mRNA in vivo and blocks translation of an mRNA molecule into the VEGF-X protein (antisensexe2x80x94Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).
Alternatively, the oligonucleotide described above can be delivered to cells by procedures in the art such that the anti-sense RNA and DNA may be expressed in vivo to inhibit production of VEGF-X in the manner described above.
Antisense constructs to VEGF-X, therefore, may inhibit the angiogenic activity of VEGF-X and prevent the further growth of or even regress solid tumours, since angiogenesis and neovascularization are essential steps in solid tumour growth. These antisense constructs may also be used to treat rheumatoid arthritis, psoriasis and diabetic retinopathy which are all characterized by abnormal angiogenesis.
A further aspect of the invention provides a host cell or organism, transformed or transfected with an expression vector according to the invention. The host cell or organism may advantageously be used in a method of producing VEGF-X, which comprises recovering any expressed VEGF-X from the host or organism transformed or transfected with the expression vector.
According to a further aspect of the invention there is also provided a transgenic cell, tissue or organism comprising a transgene capable of expressing VEGF-X protein according to the invention. The term xe2x80x9ctransgene capable of expressionxe2x80x9d as used herein means a suitable nucleic acid sequence which leads to expression of VEGF-X or proteins having the same function and/or activity. The transgene, may include, for example, genomic nucleic acid isolated from human cells or synthetic nucleic acid, including DNA integrated into the genome or in an extrachromosomal state. Preferably, the transgene comprises the nucleic acid sequence encoding the proteins according to the invention as described herein, or a functional fragment of said nucleic acid. A functional fragment of said nucleic acid should be taken to mean a fragment of the gene comprising said nucleic acid coding for the proteins according to the invention or a functional equivalent, derivative or a non-functional derivative such as a dominant negative mutant, or bioprecursor of said proteins. For example, it would be readily apparent to persons skilled in the art that nucleotide substitutions or deletions may be used using routine techniques, which do not affect the protein sequence encoded by said nucleic acid, or which encode a functional protein according to the invention.
VEGF-X protein expressed by said transgenic cell, tissue or organism or a functional equivalent or bioprecursor of said protein also forms part of the present invention.
Antibodies to the protein or polypeptide of the present invention may, advantageously, be prepared by techniques which are known in the art. For example, polyclonal antibodies may be prepared by inoculating a host animal, such as a mouse or rabbit, with the polypeptide according to the invention or an epitope thereof and recovering immune serum. Monoclonal antibodies may be prepared according to known techniques such as described by Kohler R. and Milstein C., Nature (1975) 256, 495-497. Advantageously, such antibodies may be included in a kit for identifying VEGF-X in a sample, together with means for contacting the antibody with the sample.
Advantageously, the antibody according to the invention may also be used as a medicament or in the preparation of a medicament for treating tumours or other diseases associated with expression of VEGF-X.
The invention also further provides a pharmaceutical composition comprising said antibody together with a pharmaceutically acceptable carrier diluent or excipient therefor.
Proteins which interact with the polypeptide of the invention may be identified by investigating protein-interactions using the two-hybrid vector system first proposed by Chien et al., (1991) Proc. Natl. Acad. Sci. USA 88:9578-9582.
This technique is based on functional reconstitution in vivo of a transcription factor which activates a reporter gene. More particularly the technique comprises providing an appropriate host cell with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, expressing in the host cell a first hybrid DNA sequence encoding a first fusion of a fragment or all of a nucleic acid sequence according to the invention and either said DNA binding domain or said activating domain of the transcription factor, expressing in the host at least one second hybrid DNA sequence, such as a library or the like, encoding putative binding proteins to be investigated together with the DNA binding or activating domain of the transcription factor which is not incorporated in the first fusion; detecting any binding of the proteins to be investigated with a protein according to the invention by detecting for the presence of any reporter gene product in the host cell; optionally isolating second hybrid DNA sequences encoding the binding protein.
An example of such a technique utilises the GAL4 protein in yeast. GAL4 is a transcriptional activator of galactose metabolism in yeast and has a separate domain for binding to activators upstream of the galactose metabolising genes as well as a protein binding domain. Nucleotide vectors may be constructed, one of which comprises the nucleotide residues encoding the DNA binding domain of GAL4. These binding domain residues may be fused to a known protein encoding sequence, such as for example, the nucleic acids according to the invention. The other vector comprises the residues encoding the protein binding domain of GAL4. These residues are fused to residues encoding a test protein. Any interaction between polypeptides encoded by the nucleic acid according to the invention and the protein to be tested leads to transcriptional activation of a reporter molecule in a GAL-4 transcription deficient yeast cell into which the vectors have been transformed. Preferably, a reporter molecule such as xcex2-galactosidase is activated upon restoration of transcription of the yeast galactose metabolism genes.
A further aspect of the present invention also provides a method of identifying VEGF-X in a sample, which method comprises contacting said sample with an antibody according to the invention and monitoring for any binding of any proteins to said antibody. A kit for identifying the presence of VEGF-X in a sample is also provided comprising an antibody according to the invention and means for contacting said antibody with said sample.
VEGF-X may be recovered and purified from recombinant cell cultures by methods known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography.
The VEGF-X protein of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated.
VEGF-X is particularly advantageous as a wound healing agent, where, for example, it is necessary to re-vascularize damaged tissues, or where new capillary angiogenesis is important. Accordingly, VEGF-X may be used for treatment of various types of wounds such as for example, dermal ulcers, including pressure sores, venous ulcers, and diabetic ulcers. In addition, it can be used in the treatment of full-thickness burns and injuries where angiogenesis is desired to prepare the burn in injured sites for a skin graft and flap. In this case, VEGF-X or the nucleic acid encoding it may be applied directly to the wound. VEGF-X may be used in plastic surgery when reconstruction is required following a burn, other trauma, or even for cosmetic purposes.
An important application of VEGF-X is to induce the growth of damaged bone, periodontium or ligament tissue. For example, it may be used in periodontal disease where VEGF-X is applied to the roots of the diseased teeth, leading to the formation of new bore and cementum with collagen fibre ingrowths. It can be used for regenerating supporting tissues of teeth, including alveolar bone, cementum and periodontal ligament, that have been damaged by disease and trauma.
Since angiogenesis is important in keeping wounds clean and non-infected, VEGF-X may be used in association with surgery and following the repair of cuts. It should be particularly useful in the treatment of abdominal wounds where there is a high risk of infection.
VEGF-X can also be used for the promotion of endothelialization in vascular graft surgery. In the case of vascular grafts using either transplanted or synthetic material, VEGF-X may be applied to the surface of the graft or at the junction to promote the growth of the vascular endothelial cells. One derivation of this is that VEGF-X can be used to repair the damage of myocardial and other occasions where coronary bypass surgery is needed by stimulating the growth of the transplanted tissue. Related to this is the use of VEGF-X to repair the cardiac vascular system after ischemia.
The protein of the present invention may also be employed in accordance with the present invention by expression of such protein in vivo, which is often referred to as xe2x80x9cgene therapyxe2x80x9d.
Thus, for example, cells such as bone marrow cells may be engineered with a polynucleotide (DNA or RNA) encoding for the protein ex vivo as defined herein, the engineered cells are then provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, calls may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding for the protein of the present invention.
Similarly, cells may be engineered in vivo for expression of the protein in vivo, for example, by procedures known in the art.
A further aspect of the invention comprises a method of treating a disorder mediated by expression of a protein according to the invention, by administering to a patient an amount of an antisense molecule as described herein, in sufficient concentration to alleviate or reduce the symptoms of said disorder.
Compounds which inhibit or enhance angiogenesis may be identified by providing a host cell or organism according to the invention or a transgenic cell, tissue or organism according to the invention, contacting a test compound with said cell, tissue or organism and monitoring for the effect of said compound compared to a cell tissue or organism which has not been contacted with said compound. These compounds may themselves be used as a medicament or included in a pharmaceutical composition for treatment of disorders mediated by inappropriate vascularisation or angiogenic activity.
The present inventors have also, advantageously, identified in the sequence encoding the VEGF-X protein a CUB domain, which has heretofore not previously been identified in VEGF-type growth factors. The VEGF-X protein may therefore exert dual regulatory effects via interaction with the VEGF tyrosine kinase receptors or with neuropilin receptors mediated by the CUB domain. Thus, the sequence encoding said CUB domain may be included in an expression vector for subsequent transformation of a host cell, tissue or organism.
VEGF-X or fragments thereof may be able to modulate the effects of pro-angiogenic growth factors such as VEGF as indicated in the findings presented in the examples below that the N-terminal part of the VEGF-X protein, a CUB-like domain, is able to inhibit VEGF-stimulated proliferation of HUVECs. VEGF-X or fragments thereof may therefore be useful in therapy of conditions involving inappropriate angiogenesis. Inhibition of the angiogenic activity of VEGF has been linked with inhibition of tumour growth in several models eg Kim K. J. et al, Nature 362:841-844, (1993). Additionally, agents able to inhibit angiogenesis would be expected to be useful in treating other angiogenesis-dependent diseases such a retinopathy, osteoarthritis and psoriasis (Folkman, J., Nature Medicine 1:27-31, (1995).
As identified in more detail in the Examples described herein the present inventors have surprisingly identified that the CUB domain of VEGF-X is able to inhibit stimulation of proliferation of HUVECs induced by either VEGF or bFGF. The CUB domain may, therefore, be utilised as a therapeutic agent for inhibition of angiogenesis and for treatment of condition associated with inappropriate vascularisation or angiogenesis.
Therefore according to a further aspect of the invention there is provided a method of inhibiting angiogenic activity and inappropriate vascularisation including formation and proliferation of new blood vessels, growth and development of tissues, tissue regeneration and organ and tissue repair in a subject said method comprising administering to said subject an amount of a polypeptide having an amino acid sequence from position 40 to 150 of the sequence illustrated in FIG. 10 or a nucleic acid molecule encoding the CUB domain according to the invention in sufficient concentration to reduce or prevent said angiogenic activity.
Furthermore there is also provided a method of treating or preventing any of cancer, rheumatoid arthritis, psoriasis and diabetic retinopathy, said method comprising administering to said subject an amount of a polypeptide having an amino acid sequence from position 40 to 150 of the sequence illustrated in FIG. 10 or a nucleic acid molecule encoding the CUB domain according to the invention in sufficient concentration to treat or prevent said disorders.
The CUB domain may also be used to identify compounds that inhibit or enhance angiogenic activity such as inappropriate vascularisation, in a method comprising contacting a cell expressing a VEGF receptor and/or a neuropilin 1 or 2 type receptor with said compound in the presence of a VEGF-X protein according to the invention and monitoring for the effect of said compound or said cell when compared to a cell which has not been contacted with said compound. Such compounds may then be used as appropriate to prevent or inhibit angiogenic activity to treat the disorders or conditions described herein, or in a pharmaceutical composition. An antibody to said CUB domain may also be useful in identifying other proteins having said sequences.
The above plasmids were deposited at the Belgian Coordinated Collections of Microorganisms (BCCM) at Laboratorium Voor Moleculaire Biologie-Plasmidencollectie (LMBP) B-9000, Ghent, Belgium, in accordance with the provisions of the Budapest Treaty of 28 Apr. 1977.